Antifungal and antiparasitic compounds

ABSTRACT

Novel antiparasitic and antifungal compositions are disclosed. The antiparasitic and antifungal compositions are useful for human and veterinary therapy for the treatment and/or prevention of parasitic infection. Also disclosed are novel mechanisms of identifying antifungal and antiparasitic compositions by their biochemical action on lipid synthesis and/or metabolism and/or excretion.

FIELD OF THE INVENTION

[0001] Compounds are described which represent novel, efficacious, andless toxic alternatives to current antiparasitic/antifungal treatments.Compounds having action via the biochemical mechanism of inhibition oflipid synthesis and/or metabolism and/or excretion, either by direct orindirect inhibition, will have either singly or in combinationantiparasite/antifungal activity. Such compounds, in most cases, are notchemically related by structure or chemical class to each other. Thecompounds are identified as antiparasitics and/or antifungals based onmechanism of physiologic action. Data supporting “novel use” asantiparasite/antifungal compounds are given. Many compounds hereindescribed are FDA-approved and marketed for human use fornonparasitic/nonfungal indications. Thus, the human pharmacokinetics fororal absorption, elimination rates/mechanisms, and dose-related toxicityare known.

INTRODUCTION

[0002] Status of Leishmaniasis, Trypanosomiasis, and Trichomoniasis

[0003] Current drugs most frequently used to treat leishmaniasis allrequire parenteral administration, date back 40->50 years, and all havesuch severe side-effects that treatment only in a hospital setting isrecommended (Bryceson, 1968, East African Med J 45, 110-117; Bryceson,A., 1987, The Leishmaniases in Biology and Medicine, Vol II ClinicalAspects and Control, Academic Press, New York, pp. 847-907). Noantileishmanial is Food and Drug Administration (FDA) approved and thereis no chemoprophylaxis for any leishmanial disease. Topical treatmentfor leishmanial disease is not effective even for cutaneous diseaseforms because leishmaniasis is a systemic disease (Neva, et al., 1997,Trans R Soc Trop Med Hyg 91, 473-475). There is no general vaccine forleishmaniases, although a live vaccine is used in the Middle East forcertain Leishmania (Leishmania) tropica/Leishmania (Leishmania) major toprevent facial scarring. Drug resistance is so severe in certain endemicregions that thousands are dying in India of untreatable, multidrugresistant visceral leishmaniasis; and in Northern Africa as a result ofmalnutrition exacerbated disease (Cerf, et al., 1987, J Inf Dis 156,1030-1033; de Beer, et al., 1991, Am J Trop Med Hyg 44, 283-289; Sundar,1997, Acta Parasitol Turicica 21, suppl 1, 128).

[0004] Immunodeficiency, either as the result of leishmanial tubercular-or HIV coinfections, poses serious therapeutic difficulties asleishmanial coinfection is reported to potentiate the pathology of boththese bacterial and viral infections (Alvar, et al., 1997, ClinMicrobiol Rev 10, 298-319; Bernier R, et al., 1995, J Virol 69,7282-7285; Bryceson, 1987, supra; Faraut-Gamarelli, et. al., 1997,Antimicrob Agents Chemother 41, 827-830). Global travel and commerceresult in patients having complex disease exposure history, andtransportation of leishmanial parasites far from their anticipatedendemic regions making both diagnosis and patient management difficult(Albrecht, et al., 1996, Arch Pathol Lab Med 120, 189-198).Leishmaniases have an annual incidence of 2-3 million new cases per yearwith 12 million infected and 350 million at risk in 88 countriesworldwide (Croft, 1988, Trends Pharmacol Sci 9, 376-381; World Report onTropical Diseases, 1990). The need for a orally administeredantileishmanial of low toxicity is critical.

[0005] Two major groups of diseases caused by flagellate protozoa areAfrican sleeping sickness (Trypanosoma brucei spp.) and trichomoniasis(Trichomonas/Tri trichomonas) exhibited as trichomoniasis vaginalis andtrichomoniasis foetus.

[0006] African trypanosomiasis affects both domestic and wild animals aswell as humans in mainly rural settings (Kuzoe, 1993, Acta Tropica 54,153-162; World Health Organization (WHO), 1995, Tropical DiseaseResearch, Twelfth Programme Report, Geneva Switzerland) whiletrichomoniasis is a cosmopolitan disease in men as well as women, and athreat to cattle breeding in most agricultural areas of the world(Hammill, 1989, Obstet Gynecol Clin North Am 16, 531-540; Levine, 1985,Veterinary Protozoology. Iowa State Univ. Press, Ames, pp 59-79).Treatment of the organisms causing these diseases presents problems, inpart, due to the toxicity of existing agents, and the development ofresistance to existing drugs (Kuzoe, 1993, supra; Lossick, 1989,Trichomonads Parasite in Humans. Springer-Verlag, New York, pp 324-341).

[0007] African trypanosomiasis is endemic in over 10 million squarekilometers of sub-Saharan Africa, affecting humans and all domesticatedlivestock (WHO, 1995, supra). There are an estimated 25,000 new cases ofhuman disease yearly and an animal incidence of 250-300,000 cases butthese estimates are low, based on recent civil unrest and lapses inlocal tsetse fly control and medical surveillance (WHO, 1995, supra).The primary drugs for human and veterinary trypanosomiasis have been inuse for >50 years. Resistance is spreading, especially to the onlyavailable agent for late stage central nervous system (CNS) humandisease, melarsoprol (van Nieuwenhove, 1992, Ann Soc Belg Med Trop 72,39-51; Kuzoe, 1993, supra). Melarsoprol is also toxic, with a 3-5%incidence of cerebral episodes reported (Pepin and Milord 1994, AdvParasitol 33, 2-47; Wery, 1994, Int j Antimicrob Agents 4, 227-238).Veterinary trypanocides include diminazene (Berenil®) and isometamidium(Samorin®) which are used prophylactically for control of disease incattle herds (WHO, 1995, supra; Kaminsky et al., 1993, Acta Tropica 54,19-30). Resistance to both agents has been documented in field studies(Kuzoe, 1993, supra; Schoenfeld et al., 1987, Trop Med Parasitol 38,117-180; Williamson, 1970, The African Typanosomiases. Allen & Unwin,London, pp 125-224). For these reasons, there is an urgent need todevelop new trypanocides.

[0008]Trichomonas vaginalis is one of the most prevalent sexuallytransmitted pathogen of the human urogenital tract. It infects thevaginal epithelium, causing severe irritation and the development of adischarge. In addition to social distress caused by the disease, recentevidence suggests a high incidence rate between cervical cancer andtrichomoniasis (Gram et al., 1992, Cancer Causes and Control 3,231-236). The disease is widespread, with about 3 million cases in womenannually in the United States alone (Hammill, 1989, supra). Chemotherapyfor human trichomoniasis relies on a group of 5′-nitroimidazoles, withmetronidazole (Flagyl®) being the most utilized. In the United States,metronidazole is the only available agent, although other derivativesare used in Europe and other areas. Since metronidazole has been incontinuous use since 1955, there has been increasing reports ofmetronidazole-resistant vaginitis (Meingassner & Thurner, 1979,Antimicrob Agents Chemother 15, 254-258; Wong et al., 1990,Australia-New Zealand J Obstet Gynecol 30, 169-171; Voolman & Boreham,1993, Med J Australia 159, 490). Because of its potential to producefree radicals upon reduction, it is potentially mutagenic and not givento pregnant women (Lossick, 1989, supra). At present, there is noalternative to the 5′-nitroimidazoles for therapy ofmetronidazole-refractory disease, nor for treatment of pregnant women.

[0009] Trichomonas foetus is the agent of bovine trichomoniasis, causingreproductive failure. Parasites are spread by infected bulls, multiplyin the vagina and invade the cervix and uterus. One to 16 weeks afterbreeding, abortion of the fetus occurs (Levine, 1985, supra). If theplacenta and fetal membranes are eliminated following abortion, the cowmay spontaneously recover. If some of these tissues remain inside theanimals, permanent sterility may result. There is no satisfactorytreatment for diseased cows, while treatment of bulls is tedious andexpensive. Aminoquinuride (Surfen®) or acriflavine (Trypaflavine®) maybe used topically, with dimetridazole injected into the urethra. Unlessthe bull is valuable, it is usually destroyed (Levine, 1985, supra). Thedisease is common in open range breeding ranches and may reach epidemiclevels. In Australia, 40-65% of cattle were reported to be infected,while the, prevalence in California was reported to be 14% (Yule et al.,1989, Parasitol Today 5. 373-377). The economic losses due to bovinetrichomoniasis have been estimated to be $665/infected dairy cow, whilethe widespread prevalence of the disease would account for tens ofmillions of dollars annually (Yule et al., 1989, Parasitol Today 5,373-377). The overall situation for chemotherapy of trichomoniasistherefore, is the reliance on a single drug as drug class forchemotherapy of human disease, and no effective control measures forbovine trichomoniasis.

SUMMARY OF THE INVENTION

[0010] Preliminary evidence from our ethnomedical and ethnobotanicaldrug discovery research as well as background literature describingdifferent aspects of the parasite's sterol pathway and cholesterolrequirements and importance to parasite survival, has led to thediscovery of compounds chosen on the basis of their physiologicalfunction on different parts of the sterol synthesis, and/or excretion,and/or metabolism which offer potential chemotherapeutic target(s)having low toxic potential for man. Several of these compounds have beentested for their antiparasitic/antifungal activity as described in theExamples.

[0011] The following is a brief summary of the background and data whichled to the discovery of the antiparasitic/antifungal compounds of thepresent invention.

[0012] Lipids comprise up to 15% of the total dry weight of Leishmaniaspp. (Meyer and Holz, 1966, J Biol Chem 241, 5000-5007; Beach, et al.,1979, J Parasitol 65, 203-216; Fish, et al., 1981, Mol Biochem Parasitol3, 103-116). Lipid metabolism is critical to parasite membranetransport, cell replication, and, therefore, to survival. The lipidmetabolism of Leishmania spp. including precursors, synthetic pathways,regulator molecules, and end products for membrane fatty acids, lipids,and sterols is known to mimic parts of fungal, bacterial-, plant-, andhuman lipid pathways, while completely duplicating none. Becauseleishmanial lipid metabolism is unique among organisms, geneticallyconserved (Wendt, et al., 1997, Science 277, 1811-1815), andbiochemically-tightly regulated (Thompson, 1992, The Regulation ofMembrane Lipid Metabolism. CRC Press, Ann Arbor, pp 230), the sterolpathway has the potential to provide us chemotherapeutic targets notduplicated in humans (drug development).

[0013] Leishmania share with plants (and animals) that they rely onmevalonic acid as a precursor for de novo sterol synthesis (Holz, 1985,Leishmaniasis. Elsevier, N.Y., pp 79-92; Thimann, 1977, Hormone Actionin the Life of Plants. University of Massachusets press, Amherst, pp.448; Thompson, 1992, supra) However, the major sterol of leishmanial andfungal membranes, synthesized de novo by these parasites, is notcholesterol (like humans), but a 24-substituted sterol (ergosterol orepisterol or provitamin D2). Ergosterol is synthesized by theseparasites de novo from acetylCoA, to mevalonate, to squalene, tolanosterol, and 4 steps later to ergosterol (Holz, 1985, supra). Coppensand Courtoy (1995, Mol Biochem Parasitol 73, 179-188) showed thatprocyclics of T. brucei normally contain ergosterol synthesized de novo,a pathway shared with Leishmania.

[0014] However, Leishmania require cholesterol. Unlike man, but likeclosely related Kinetoplastid parasites, of the genus Trypanbsoma,Leishmania “salvage” cholesterol from their environment, i.e., frommacrophages and monocytes (the LDL/cholesterol plasma clearance cells)in the mammalian reticuloendothelial system. Free cholesterol and freefatty acids do not occur normally in plasma. The cholesterol esters offatty acids, which are by themselves insoluble in plasma, are located inthe low density lipoprotein, LDL, as a nonpolar core surrounded with apolar shell of phospholipids, apoprotein, and unesterified cholesterol,thus ensuring solubilization and transport (Ormerod & Venkatesan, 1982,Microbiol Rev 46, 296-307; Thompson, 1992, supra). Leishmania reside inmononuclear macrophages, which comprise the major part of low-densitylipoprotein (LDL) plasma clearance system via both receptor andreceptor-independent mechanisms (Goldstein & Brown, 1976, Curr Top CellRegul 11, 147-181; 1977, Ann Rev Biochem 46, 897-930; Weisgraber, etal., 1978, J Biol Chem 253, 9053-9062; Pangburn, et al., 1981, J BiolChem 256, 3340-3347; Bilheimer, et al, 1982, Proc Natl Acad Sci USA 79,3305-3309; Haughan, et al., 1992, Biochem Pharmacol 44, 2199-2206).Transport of LDL-cholesterol via either or both mechanisms into infectedmonocytes would thus allow leishmanial parasites to meet theircholesterol requirement. Drugs which interrupt the quantity, transport,or delivery of cholesterol to the parasite would have potential toadversely affect leishmanial survival.

[0015] There are marked metabolic similarities between leishmanial andtrypanosomal lipid acquisition and metabolism. Bloodstream forms ofTrypanosoma brucei spp. can ingest particulate fat (Wooten & Halsey,1957, Parasitol 47, 427-431), and, like Leishmania, Trypanosoma bruceirhodesiense depends on the cholesterol of their habitat (Dixon et al.,1972, Comp Biochem Physiol 41B, 1-18).

[0016] Coppens and colleagues (1995, Mol Biochem Parasitol 73, 179-188)showed that the enzyme inhibitor, synvinolin (simvastatin or Zocor®),potentiates growth inhibition of Trypanosoma brucei in the presence ofdrugs interfering with the exogenous supply of cholesterol; andconversely, growth inhibition by synvinolin can be reversed by LDL,mevalonate, squalene or cholesterol. Coppens and Courtoy (1995, supra)showed that procyclics of T. brucei spp. normally incorporate exogenouscholesterol in their membranes. These investigators further demonstratedthat growth of the culture-adapted trypanosomes is accelerated bysupplementation of the medium with low density lipoprotein (LDL)particles which were endocytosed by the parasites via areceptor-mediated mechanism.

[0017] We observed that traditional medical herbal therapies, containingplant sterols having the cholestane backbone but with hydrophillicsubstitutent side chains, first destabilized then killed parasites invitro in a dose-dependent manner. Chemical analyses of the structure ofthe antiparasitic active moieties from these plants (>70 tested) mostfrequently revealed an isoprenoid, terpenoid, or steroidal structureresembling but not duplicating normal mammalian sterolgenic precursors.It is known, as previously discussed, that Leishmania spp. and AfricanTrypanosoma spp. take up cholesterol and any cholestane-backbonemolecule (Dixon, et al., 1972, supra; Haughan, et al. 1995, supra). Webelieve that substitute “plant cholesterol-like” molecules serve todestabilize parasites' membranes because of either addition of newhydrophillic sidegroups; or replacement of typically hydrophobicside-groups with more hydrophillic side-groups. These observations, inaddition to the knowledge of the importance of cholesterol andcholesterol synthesis in the organism, appeared to validate the use ofthese medicinal plants as herbal remedies for treatment of protozoanparasitic infections.

[0018] Therefore, at several points within the sterol synthesis andcholesterol salvage pathways, we have identified molecules chemically orfunctionally similar to the natural component, but which act toshut-down leishmanial function.

[0019] Therefore, it is one object of the present invention to provide anovel method for identifying compounds having antiparasitic andantifungal activity based on the physiological action of the compoundsin the sterol synthesis and/or metabolism, and/or excretion pathway ofthe parasite.

[0020] It is also an object of the present invention to provide a novelmethod for identifying antifungal and antiparasitic compounds by theirability to inhibit cholesterol synthesis and/or metabolism and/orexcretion, directly or indirectly.

[0021] It is further an object of the present invention to provide novelantiparasitic and antifungal agents which are capable of oraladministration, and are efficacious and less toxic alternatives toagents heretofore used for the treatment of fungal and/or parasiticinfection in humans and animals.

[0022] A still further object of the present invention is to provide anovel method of using existing compounds not previously known to haveantifungal or antiparasitic activity for the prevention and/or treatmentof fungal or parasitic infection in humans and animals.

[0023] It is also an object of the present invention to provideantiparasitic and antifungal compositions for either prophylactic orfield treatment.

[0024] A further object of the present invention includes the combinedtherapy that can be obtained by treating patients with leishmania,trichomoniasis, or trypanosomiasis, with a combination of the compoundsof the present invention, preferbly the combination is chosen such thatcompounds which inhibit different parts of the cholesterol pathway arecombined.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] These and other features, aspects, and advantages of the presentinvention will become better understood with reference to the followingdescription and appended claims, and accompanying drawings where:

[0026]FIG. 1. A schematic representation of the mechanism of cholesterolregulation indicating eleven types of inhibitors of lipid metabolism,synthesis, or excretion having antiparasitic/antifungal properties (boldcap letters).

DETAILED DESCRIPTION

[0027] Forty-two medicinal plants were identified as havingantileishmanial properties from ethnomedical studies and eitherantileishmanial/antifungal properties from ethnobotanical research.Fifty percent (21/42) plants and 59/121 extracts tested showed in vitroantileishmanial activity. The chemical isolation strategy focusedpreferentially on isolation of di- and tri-terpenes (sterol-like)compounds which seemed to contain highly active (>90% cidal in vitro)antileishmanial compounds. The first compound to be characterized was aspirostanol saponin, Mannispirotan A, isolated from the fruit pulp ofDracaena manii (Okunji et al., 1990, Int J Crude Drug Res 28, 193-199)Study of the structure (shown below at ‘A’) revealed a resemblance to asterol nuclei structure.

Spirostanol Saponin

[0028] Four additional highly active extracts have been purified andtheir structures, which include more than 25 separate compounds,determined. Most are compounds that have chemical congeners,isoprenoids, di- and triterpenoids common to lipid metabolism; a few areberberine-like or -dimers presented in U.S. Pat. No. 5,290,553, to Iwu,et al., 1994. All documents cited herein supra or infra are incorporatedin their entirety by reference thereto. Knowledge of structure activityrelationship (SAR) has allowed us to formulate hypotheses for themechanism of antiparasite physiologic inhibition.

[0029] When additional plant extracts were examined, and additionalactive structures elucidated, namely, Sakuretin from Eupatoriumodoratum, Labdane-dial from Aframomum danielli, and Afromomumaulocacaxpus, unexpectedly, the structures of these compounds did notresemble cholesterol, but instead resembled Vitamin D2 and possibleparts of a squalene isoprenoid structure as it is cyclized.

Labdane-Dial from Aframomum danielli

[0030]

Sakurentin from Eupatorium odorantum

[0031] As discussed previously, the parasite can synthesize ergosterol(Holz, 1985, supra), also known as pro-vitamin D2 (structure shownbelow) but they require cholesterol which cannot be synthesized by theorganism, and therefore, has to be salvaged from the host. Whenleishmania infects a host, within minutes, the organism localizes to theliver, and it is in the liver that host ergosterol (provitamin D) isconverted to vitamin D₂. The conversion of ergosterol to cholesterolcauses an increase in Ca⁺⁺ ion concentration. It had been reportedpreviously that the ability of macrophages to kill leishmania is reducedunder increased Ca⁺⁺ conditions (Olivier, 1996, Parasitol Today 12,145-150).

[0032] The structures of the active ingredients in the medicinal plants,and the fact that the parasites must have to scavenge cholesterol, madeus focus on the cholesterol synthesis pathway as described in FIG. 1.

[0033] We have found that drugs known to inhibit different parts of thecholesterol pathway can be, for the first time, used as antiparasiticagents. This discovery was novel and unexpected and was the result ofputting together several different disparate pieces of evidence. None ofthe drugs discussed in this application were used or suggested for thetreatment of leishmania, trypanosomiasis, or trichomoniasis. It is onlyafter the elucidation of the chemical structure of the active compoundsin the medicinal plants in addition to inventive activity that therelationship between the sterol pathway and possible antiparasiticagents was discovered. Most are human-use, FDA-approved drugs foralternative medical indications.

[0034] Our initial work focused on the following metabolic steps ofleishmanial steroid metabolism which we have ascertained are criticalfor parasite survival: (1) butyric acid as a required precursor for bothfatty acid and sterol synthesis; (2) mevalonic acid synthesis fromacetylCoA; (3) squalene synthesis from mevalonic acid; (4) ergosterolsynthesis from lanosterol; and (5) sterol (cholestane-analog uptake).

[0035] At each step and in each category of inhibitory compounds,suitable examples of drugs which may be used as antiparasitic/antifungalagents are mentioned. However, these examples are not meant to belimiting, and it is understood that other suitable drugs, known or to bediscovered, which belong in the categories mentioned can be assayed andused as antiparasitic/antifungal agents. The assays for testing whetheror not a drug is antiparasitic/antifungal are known, one of which isdescribed in the Examples below.

[0036] Butyrate Inhibition

[0037] Butyrate is a key fatty acid precursor of acetyl-CoA. Acetyl-CoAand free fatty acids are critical to eukaryotic cells' energy productionvia beta-oxidation. Fatty acids are activated to acetyl-CoA derivatives,transported into the matrix of the mitochondria via the carnitine cycle,where they undergo beta-oxidation (Murray et al., 1988, Harper'sBiochemistry, 21st ed., Appleton and Lange, Publ., Norwalk, Conn.).Beta-oxidation of fatty acids results in the reduced coenzymes FADH2 andNADH. The oxidation of 1 mole of FADH2 yields 2 moles ATP, and theoxidation of 1 mole of NADH yields 3 moles of ATP. From work in ourlaboratory, we know that butyrate is a key factor for leishmanialmetabolism. Using ¹⁴C-labelled butyrate, we showed that it is readilytaken up and rapidly metabolized to 14CO₂ by Leishmania spp. (Jackson,et al., 1989, Am J Trop Med Hyg 41, 318-330; Jackson, et al., 1990, Am JTrop Med Hyg 43, 464-480). Any compound comprising a butyrate inhibitorcan be used as an antiparasitic/antifungal agent. Suitable forms of suchcompounds are cefaloglycin and xenbucine. Cefaloglycin reduces oxidationand uptake of butyrate. Cefaloglycin,7-(2-amino-2-phenylacetamido)-3-(hydroxymethyl)-8-oxo-5-Thia-1-azabicyclo[4.2.0]acetate(ester), chemical registration no. 3577-01-3, or aminophenylacetamidocephalosporanic acids, are known in the art and marketed under the nameKafocin® by Eli Lilly and Co. Indianapolis, Ind. A process for theirproduction is described in U.S. Pat. No. 3,422,103 to Wilfred et al.,Jan. 14, 1969, herein incorporated in its entirety. Xenbucin,2-(4-biphenyl)butyric acid; alpha-ethyl-[1,1′-biphenyl]-4-acetic acid,chemical ID no. 959-10-4, described in Brit. 1,168,542 (1969, Maggioni),preparation described in U.S. Pat. No. 4,542,233 to Piccolo et al.,September, 1985, marketed under the name Liosol® by MaggioniPharmaceutici, Italy.

[0038] CHOLINE: Choline is the starting material for lipogenesis viaproduction of acetyl-CoA. Dapsone (4,4′-diaminodiphenyl sulfone) hasbeen reported active against human leishmaniasis via choline inhibition(Dogra, 1992, Infection 20, 189-191). This drug is believed to act viaparaminobenzoic (PABA) acid-reversible block of the folic acidmetabolism of parasitic protozoa. It is unlikely that this is themechanism by which dapsone functions against Leishmania.

[0039] Leishmania rely exclusively on salvage mechanisms for purinesynthesis and metabolism. Presumably, a dapsone block of purinesynthesis via prevention of the reduction of folic acid to thetetrahydro-derivative and, thus, transport of the formyl carbon into thepurine ring (positions 2 & 8 of purine), could not occur in leishmanialparasites utilizing preformed purines to synthesize nucleic acids andlacking these de novo synthetic pathways Likewise, a thymidylatesynthetase block is unlikely to prove fatal, since Leishmania salvage aswell as synthesize pyrimidines.

[0040] A choline inhibitory pathway for antileishmanial activity (assuggested by Dogra, 1991, Trans R Soc Trop Med Hyg 85, 212-213; Dogra,1992, supra) is more likely, although the mechanism of such inhibition,is a more complex problem to investigate. Dogra (1991, supra;1992,supra) postulated that dapsone probably acts against Leishmania byinhibition of choline incorporation into lecithin in the cell membrane,thus decreasing phospholipid synthesis. It is the relationship ofcholine inhibition to other drug-sensitive lipid metabolic target(s)that we wish to investigate therapeutically.

[0041] Dapsone has an IC₅₀ of 600 mM (1.49 mg/ml) in vitro againstLeishmania major promastigotes in a chemically defined medium. Dapsoneinhibition was not reversible by p-aminobenzoate (PABA) folate orthymidine (Peixoto and Beverley, 1987, Antimicrob Agents Chemother 31,1571-1578). Invanetich and Santi (1990a, FASEB J 4, 1591-1597) notedthat: “Antifolates commonly used to treat microbial infections are poorinhibitors of Leishmania major dihydrofolate reductase.” Peixoto andBeverley (1987, supra) concluded that “the mode of action of sulfa drugs[dapsone] is not by the classical route of de novo folate synthesis”.These results with dapsone inhibition are understandable based onprevious work on the folate metabolism of these protozoan parasites.

[0042] Clofazimine,N,5-Bis(4-chlorophenyl)-3,5-dihydro-3-[(methylethyl)imino]-2-phenazinamine;3-(p-choroanilino)-10-(p-chlorophenyl)-2,10-dihydro-2-(isopropylimino)phenazine,chemical registration no. 2030-63-9, marketed as Lamprene®, ananticancer and antimycobacterial riminophenazine drug, is active viaphospholipase A2-mediated oxidative and nonoxidative mechanisms(Arunthathi and Satheesh 1997, Lepr Rev 68(3), 233, 241; Ruff et al.,1998, Ann Oncol 9, 217-219: van Rensburg, et al., 1993, Cancer Res 53,318-323; Venkastesan, et al., 1997, Lepr Rev 68, 242-246).Antimycobacterial dose is 50 mg/day or 100 mg on alternate days(Venkastesan, et al., 1997, supra). Riminophenazine drugs have neverbeen used or proposed as antileishmanial/antitrypanosomals. Human doserecommended are 100-200 mg/day, although doses 400 mg-600 mg/day can begiven.

[0043] Other suitable examples of inhibitory compounds includeeldacimibe,1,3-Dioxane-4,6-dione,5-[[[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]amino][[[4-(2,2-dimethylprophyl)phenyl]-methyl]hexylamino]methylene]-2,2-dimethyl-;(2)Cyclicisopropylidene[(3,5-di-tert-butyl-4-hydroxyanilino)[hexyl(p-neopentyl-benzyl)amino]methylene]malonate,chemical registration no. 141993-70-6, marketed as Eldacimibe® byWyeth-Ayerst Laboratories, Philadelphia, Pa., and lecimibide, Urea,N′-(2,4-difluorophenyl)-N-(5-((4,5-diphenyl-1H-imidazol-2-yl)thio-)pentyl)-N-heptyl-,chemical registration no. 130804-35-2, marketed as Lecimibide® by MerchPharmaceutical Co., Whitehouse Station, N.J.

[0044] Squalene

[0045] Some compounds act indirectly on the Leishmania as steroidalsynthesis regulators: human insulin, human transferrin, and low densitylipoprotein (LDL). Both transferrin and insulin are either inhibitors orgrowth stimulants of human and, possibly also, leishmanial sterolsynthesis depending on concentration (Schroepfer, 1981, Ann Rev Biochem50, 585-621; Thompson, 1992, The Regulation of Membrane LiDidMetabolism. CRC Press Ann Arbor, pp 230; Jackson, et al., 1989, supra).Other sterols, synthesized only by the Leishmania and fungi, may act toregulate the host cells (monocyte or macrophage) to prevent parasitekilling, e.g. by increasing intracellular Ca⁺⁺ level (Oliver, 1996,supra). Sacchettini and Poulter (1997, Science 277, 1788-1789) notedthat the isoprenoids, or steroidal building blocks, are a remarkablydiverse chemical class comprising over 23,000 individual compounds. Forover 100 years, dating back to traditional medicine, it has been knownmany antifungals also sometimes have antiparasitic properties (reviewed,Steck, 1972, The Chemotherapy of Protozoan Diseases, Vol II, p 7.61-7.63and 11.100-110, U.S. Government Printing Office, Washington, D.C.,#O-462-576). Additionally, it has been known for over 50 years thatantifungals such as amphotericin B, pentamidine, and ketoconazole (Neal,1987, The Leishmaniases in Biology and Medicine, Vol II Clinical Aspectsand Control. Academic Press, New York, pp. 793-845) have antileishmanialactivity. Lipid analyses of several Leishmania spp. revealed that theseparasites' membranes contain a high percentage of ergosterol, a sterolmost frequently found in fungi and some bacteria (Holz, 1985, supra)which presents a basis for common mechanism of action of antifungaldrugs on leishmania. Terbinafine is recognized as an clinical antifungaland cutaneous antibacterial (Back, et al., 1992, Brit J Dermatol 126(Suppl 39), 14-18; Baudraz-Rosselet et al., 1992, Brit J Dermatol 126(Suppl 39), 40-46; Finlay, 1992, Brit J Dermatol 126 (Suppl 39), 28-32;Goodfield, 1992, Brit J Dermatol 126 (Suppl 39), 33-35; Hay andStratigos, 1992, Brit J Dermatol 126 (Suppl 39), 1-69; Haroon, et al.,1992, Brit J Dermatol 126 (Suppl 39), 47-50; Hull and Vismer, 1992, BritJ Dermatol 126 (Suppl 39), 51-55; Kovarik, et al., 1992, Brit J Dermatol126 (Suppl 39), 8-13; Nolting and Brautigam, 1992, Brit J Dermatol 126(Suppl 39), 56-60; Roberts, 1992, Brit J Denmatol 126 (Suppl 39), 23-27;Ryder, 1992, Biochem J 230, 765-770; Van der Schroeff, et al., 1992,Brit J Dermatol 126 (Suppl 39), 36-39; Villars and Jones, 1992, Brit JDermatol 126 (Suppl 39), 61-69).

[0046] Recent antiparasite investigations of known antifungals haveprimarily involved the combination of known antileishmanials with one ormore newer antifungals, the latter to include the squalene oxidaseinhibitor, terbinafine. The antifungal terbinafine has shown preliminaryantitrypanosomal activity in vitro and in primary rodent drug screeningsystems against Trypansoma cruzi, the etiologic agent of Chagas' disease(Urbina et al., 1996, Science 273, 969-971) and Leishmania mexicana, 2cutaneous leishmanial subspecies (Goad et al., 1985, Biochem Pharmacol34, 3785-3788; Berman and Gallalee, 1987, J Parasitol 73, 671-673).

[0047] Complex structure activity relationship (SAR) studies ofsynthetic and natural product (biologically derived) squalene synthetaseand squalene oxidase inhibitors have shown several such compounds havein vitro and in vivo activities having human hypocholesteremicpotential. Abe and collegues (1994, supra) reviewed SAR data from 284squalene synthesis inhibitors. Selected data from a few of the besthypocholesteric candidates (from Abe, et al, 1994 supra) follow.

[0048] Suitable examples of Squalene Synthetase inhibitors include:

[0049] 1. Thioether analog of 2,3-oxidosqualene (Abe, et al, 1994,supra; Zheng, et al, 1995, J Am Chem Soc 117, 670-680) ICC₅₀ 0.0023 uM

[0050] 2. 29-methylidene-2,3-oxidosqualene, an irreversible inhibitor ofoxidosqualene cyclase (Abe, et al, 1994, supra; Xiao and Prestwich,1991, J Am Chem Soc 113, 9673-9674)

[0051] 3. Ether analog of farnesyl diphosphate (IC₅₀ 0.05 uM, Abe, etal, 1994, supra)

[0052] 4. Farnesyl bisphosphonate (no oral activity, IC₅₀ 0.00027 uM,Abe, et al, 1994, supra)

[0053] 5. Natural product from Phoma sp. C2932, Squalestatins 1,2,3(IC₅₀ 15.2, 15.1, 5.9 nM, respectively, Abe, et al, 1994, supra)

[0054] 6. Natural products from ATCC 20986, Sporormilla intermedia, andLeptodontium elatius: Zaragozic acid A,B,C, IC₅₀ 78, 29, 45 pM ,respectively (Abe, et al, 1994, supra)

[0055] 7. CP-225,917 (Pfizer) and CP-263,114 (Pfizer), both compoundsinhibit squalene synthase and farnesylprotein transferase (Borman, 1999,Chemical and Engineeing News Jun. 7, 1999, 8-9; Service, 1999, Science284, 1598-1599; Dabrah et al., 1997, J Antibiot 50, 1-7)

[0056] Suitable examples of inhibitors of Squalene Oxidase include:

[0057] 1. Naftifine, 1-Naphthalenemethanamine,N-methyl-N-(3-phenyl-2-propenyl)-(E), chemical registration no.65472-88-0, marketed as an antifungal under Exoderil® or Naftin®, anddescribed in a patent to Berney on Aug. 4, 1981, U.S. Pat. No.4,282,251. IC₅₀ 0.93 uM (Abe et al, 1994, supra; Georgopoulis et al.,1981, Antimicrob Agents Chemother 19, 386-389; Paltauf et al., 1982,Biochim Biophys Acta 712, 268-273; Petranyi et al., 1984, Science 224,1239-1241; Ryder, 1984, In Nombel C. (ed.) Microbial Cell Wall Synthesisand Autolysis, Elsevier, N.Y., pp 313-321)

[0058] 2. Terbinafine, 1-Naphthalenemethanamine,N-(6,6-dimethyl-2-hepten-4-ynyl)-N-methyl-, (E)-, an antimycoticallylamine, chemical registration no. 91161-71-6, or turbinefinehydrochloride, chemical registration no. 78628-80-5. Turbinafine ismarketed as Lamisil®, and its preparation is described in Eur PatentAppl. no. 24,587 to A. Stutz, 1981. Terbinafine has been shown to haveactivity against Leishmania species in vitro and in animal and humanclinical trials (Abe, et al, 1994, supra; Bahamdan et al., 1997, Int JDermatol 36, 59-60; Ellenberger and Beverley, 1989, J Biol Chem 264,15094-15103; Goad, et al., 1985, Biochem Pharmacol 34, 3785-3788;Gonzales-Ruperez et al., 1997, Dermatology 194, 85-86; Rangel et al.,1996, Antimicrob Agents Chemother 40, 2785-2791; Urbina 1997,Parasitology 114 Suppl S91-S99; Vannier-Santos et al., 1995, J EukaryotMicrobiol 42, 337-346).

[0059] 3. Butenafine,N-(p-tert-Butylbenzyl)-N-methyl-1-naphthalenemethylamine, a benzyl amineantifungal, chemical registration no. 101828-21-1, or butenafinehydrochloride, chemical registration no. 101827-46-7, marketed asMentax® by Penederm Inc. Foster City, Calif. Preparation is described inU.S. Pat. No. 4,822,822 to Arita et al. on Apr. 18, 1989.

[0060] 4. SDZ 87-469 (Georgopapadakou et al., 1992, Antimicrob AgentsChemother 36, 1779-1781, and references cited therein; Ryder and Frank,19992, J Med Vet Mycol 30, 452-460) IC₅₀ 0.011 uM (Abe, et al, 1994,supra)

[0061] 5. NB-598, (Matzno et al., 1997, J lipid Res 38, 1639-1648 andreferences cited therein) IC₅₀ 0.75 nM (Abe, et al, 1994, supra)

[0062] 6. TMD, 4,4,10beta-trimethyl-trans-decal-3beta-ol (Abe, et al,1994, supra; Nelson et al, 1978, J Am Chem Soc 100, 4900-4902)

[0063] HMGCOA, 3-hydroxy-3-methylglutaryl CoA Reductase Inhibitors

[0064] Mevalonic acid, a precursor to human sterols and steroids; and inplants, to hormones and carotenoids, is available to Leishmania both viathe host human monocyte or macrophage; and within the sandfly vector, inthe bloodmeal and plant juices essential to sustain the fly (Leclercq,1969, Entomological Parasitology. Pergamon Press, New York, pp 158;Beytia and Porter, 1976, Ann Rev Biochem 45, 112-142; Thimann, 1977,Hormone Action in the Life of Plants. University of Massachusetts Press,Amherst, pp 448; Caspi, 1984, Tetrahedron 42, 3-50). Most sandflyspecies known to transmit Leishmania, require not only blood but alsoplant fluids to maintain proper hydration for survival. Avoiding lethalultraviolet rays of sunlight, sandflies rest in moist shady areas duringthe day to emerge-in the evening to feed. Plants, particularly those inthe tropical climates where sandflies are most numerous, tend to losewater in the intense heat of the afternoon. The plant hormoneresponsible for closing leaf stomata to prevent plant dehydration,abscisic acid, is made in response to water loss. Abscisic acid is knownto increase 200-fold in a dehydrated plant. Three mevalonic acidmolecules are required to produce one molecule of abcissic acid. As moreabscisic acid is required in the heat of the day, so is its precursor,mevalonic acid. Sandflies feed at twilight when plant dehydration,abscisic acid, and melvalonic acid would be expected to be near peakdaily level in tropical plants.

[0065] In animals and humans, mevalonic acid is also an importantprecursor to sterol and steroid synthesis, so would likewise beavailable to leishmanial amastigotes inhabiting the monocytes,macrophages and hepatic cells. On the basis of host and vectorphysiology alone, mevalonic acid appeared to be implicated as animportant precursor molecule for leishmanial sterolgenesis.

[0066] To investigate this, we used ¹⁴C-mevalonic acid to determine rateof incorporation by Leishmania (1.0 ng/hr/108 parasites at 25° C., usingthe respirometric assay. We also looked at mevalonic acid catabolism andfound mevalonate is sparingly metabolized to CO2 (less than 1/25 therate of aspartic acid metabolism, a most rapidly catabolized amino acid,Jackson, et al., 1989, Am J Trop Med Hyg 41, 318-330) even when thepromastigotes are maintained under starvation condition for 30 minutes.When mevalonate was added as a nutritional supplement the parasites grewprofusely but less rapidly than parallel unsupplemented controlcultures. (The amount mevalonate added to in vitro cultures wasdetermined based on incorporation rate relative to aspartic acid, andthis may have resulted in too high an estimated mevalonic acidconcentration.) However, mevalonate-fed cultures remained in logarithmicphase growth 2-fold longer (>10 days) than parallel unsupplementedcultures (which ended log phase growth at 4-5 days of culture). Giventhese preliminary observations: it appears Leishmania (a) incorporatemevalonic acid readily from their environment; (b) catabolism is sparedeven under starvation conditions; and (c) mevalonic acid can act as anutritional supplement in vitro.

[0067] Three-hydroxy-3-methylglutaryl CoA reductase is a protein of theendoplasmic reticulum whose concentration is determined by rates ofcholesterol synthesis. HMG-COA reductase catalyzes the reductivedeacylation of HMG-COA to mevalonate by two molecules of NADPH. In mosttissues this is considered the first committed step in sterol/isoprenoidbiosynthesis. In most biologic systems studied, this reaction is therate-limiting step for sterol biosynthesis (Danielsson and Sjovall,1985). Most widely used hypercholesteremic drugs have their mode ofaction at this irreversible synthetic step catalyzed by3-hydroxy-3-methylglutaryl CoA reductase (HMCoAR).

[0068] HMG-COA reductase inhibitors lower plasma total cholesterol, lowdensity lipoprotein (LDL), and B apolipoprotein in humans as the resultof decreased cholesterol synthesis and enhanced removal of LDLs via theLDL receptor pathway in hepatocytes (Hoeg and Brewer, 1987; Tolbert,1987).

[0069] No HMG-COA reductase inhibitor has ever been used or previouslytested as an antileishmanial or for South and Central AmericanTrypansoma rangeli. There are two references to anti-Trypanosoma(Schizotrypanum) cruzi, South and Central American trypanosome species,in vitro (Florin-Christensen, et al., 1990; Urbina, et al., 1993) and invivo mouse testing of mevinolin (Lovastatin®) testing, alone and incombination with ketoconazole and terbinafine (Urbina, et al., 1993).Coppens and colleagues (1995; and, Coppens and Courtoy, 1995) showedthat the enzyme inhibitor, synvinolin (simvastatin or Zocor®),potentiates growth inhibition of Trypanosoma brucei in the presence ofdrugs interfering with the exogenous supply of cholesterol; andconversely, growth inhibition by synvinolin can be reversed by LDL,mevalonate, squalene or cholesterol.

[0070] All 3-hydroxy-3-methylglutaryl CoA reductase inhibitors, orvastatins, are not a chemical class effect but vary widely betweenHMGCoA reductase inhibitors (Bocan, et al., 1994; Haruyama, et al.,1986; Kempen, et al., 1991; Nakaya, et al., 1986; Serizawa, et al.,1983; Tsujita, et al., 1986; Yoshino, et al., 1986)(e.g. atorvastatin,CI981; PD134965; pravastatin, CS-514, Eptastatin, SQ 31000; BMY22089;simvastatin, Synvinolin, MK-733; monacolin K, MB-530B; mevinolin,lovastatin; mevastatin, ML-236B, Compactin) do not have the sameefficacy for preventing atherosclerotic lesions, inhibition ofcholesterol synthesis in target tissue(s), reducing cellularaccumulation of free and/or esterified cholesterol, degradation of LDL,or synthesis of phosphotidylcholine and sphingomyelin.

[0071] This observation may be due, in part to the chemical design ofvarious vastatins, for example, pravastatin differs from other HMG-COAreductase inhibitors in two aspects. In pravastatin, the 6-position onthe decalin ring is occupied by a hydroxyl group, whereas, in lovastatinand simvastatin, this same position is occupied by a methyl group. Thisdifference in structure is responsible for the different physiochemicalproperties of these drugs and confers on pravastatin its hydrophiliccharacteristics. Lovastatin and simvastatin are hydrophobic and designedwith the objective of obtaining high levels of hepatoselectivity(Keidar, et al., 1994; Sirtori, 1993). Pravastatin is administered as asodium salt of an open acid and is the active inhibitor of HMG-CoAreductase; lovastatin and simvastatin are prodrugs and are given asinactive lactones that, following oral administration, are hydrolyzed toan active inhibitor” (Keidar, et al., 1994). Pravastatin is manufacturedby Bristol-Myers Squibb; Merck manufacturers lovastatin and simvastatin(Zurer, 1997); and Sanyo, eptastatin (Yoshino, et al., 1986).

[0072] Suitable HMG-CoA reductase inhibitors include:

[0073] 1) Pravastatin, [1S-(1-alpha(beta-S*,delta-S*),2-alpha,6-alpha,8-beta(R*),8a-alpha]]-1,2,6,7,8,8a-hexahydro-2-methyl-8-(2-methyl-1-oxobutoxy)-beta,delta,6-trihydroxy-1-Naphthaleneheptanoicacid monosodium salt, a highly selective cholesterol synthesis inhibitorof hepatic, intestinal cells (ileum), and in monocyte-derivedmacrophages (Keidar, et al., 1994). Pravastatin, chemical registrationno. 81093-37-0, marketed as Pravachol® by Bristol-Myers Squib,Wallingford, Conn. or as Eptastatin from Sanyo, as well as others. Thepreparation of pravastatin is described in U.S. Pat. No. 4,346,227 toTerahara et al., August, 1982. When humans were given a dose of 40mg/day for 8 weeks, pravastatin resulted in a dose-dependent inhibitionof macrophage cholesterol synthesis; LDL increased 119% with 0.1 mg/mlpravastatin; <or =0.19 mg/ml increased cholesterol esterification; >0.19mg/ml inhibited cholesterol esterification; pravastatin inhibitedcholesterol synthesis 55-62% and increased LDL degradation by 57%(Keidar, et al., 1994).

[0074] 2) Simvastatin, [1S-[1-alpha(beta-S*,delta-S*),2-alpha,6-alpha,8-beta(R*), 8a-alpha]]2,2-dimethylbutanoic acid1,2,3,7,8,8a-hexahydro-3,7-dimethyl-8-[2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-vl)ethyl]-1-naphthalenylester, a competitive inhibitor of HMG-COA reductase, chemicalregistration no. 79902-63-9, marketed in several forms, e.g. Zocor® fromMerck & Co., Whitehouse Station, N.J., preparation described in U.S.Pat. No. 4,444784 to Hoffman et al. April, 1984. In a longterm study ofsimvastatin (3-5.4 years) at doses 0.5 of pravastatin and 0.125 offluvastatin, simvastatin (at 10 to 40 mg/day doses) lowered serumcholesterol from baseline 20-40%; lowered low density lipoproteincholesterol 35-45%; and reduced triglycerides 10-20% (Plosker GL,McTavish D, 1995).

[0075] 3) Fluvastatin, 6-Heptenoic acid,3,5-dihydro-7-[3-(4-fluorophenyl)-1-(1-methylethyl)-1H-indol-2-yl-]-[R*,S*(E)]-, (+−)-, chemical registration nos. 93957-55-2 and 93957-54-1,marketed as Lescol® from Sandoz, East Hanover, N.J., described in U.S.Pat. No. 4,739,073, 1984. Review of Pharmacology and therapeutics use,Levy et al., 1993, Circulation 87, Suppl III-45 to III-53.

[0076] 4) Atorvastatin, 1H-Pyrrole-1-heptanoic acid,2-(4-fluorophenyl)-beta,delta-dihydroxy-5-(1-methylethyl)-3-pheny-1-4-[(phenylamino)carbonyl)-,[R-(R*,R*)]-, chemical registration nos. 134523-00-5 and 11086248-1,described in U.S. Pat. No. 5,273,995 to Roth, December 1993, marketed byWarner-Lambert, Morris Plains, N.J.

[0077] 5) Cerivastatin,7-[4-(4-fluorophenyl)-5-(methoxymethyl)-2,6-bis(1-methylethyl)-3-pyridinyl]-3,5-dihydroxy-,monosodium salt, [S-(R*,S*-(E))]]-, cerivastatin sodium, chemicalregistration no 143201-11-0, marketed as Baycol® from Bayer Corp. WestHaven, Conn.

[0078] 6) Crilvastatin, L-Proline, 5-oxo-, 3,3,5-trimethylcyclohexylester, chemical registration no. 120551-59-9, available from LaboratoirePan Medica, France.

[0079] 7) Dalvastatin,2H-Pyran-2-one,tetrahydro-6-[2-(2-(4-fluoro-3-methylphenyl)-4,4,6,6-tetramethyl--1-cyclohexen-1-yl]ethenyl]-4-hydroxy-,[4R-(4-alpha,6-beta(E)]]-, chemical registration nos. 135910-20-2,132100-551, available from Rhone-Poulenc Rore Pharmaceuticals, Inc.Collegeville, Pa.

[0080] 8) Lovastatin, Butanoic acid,2-methyl-,1,2,3,7,8,8a-hexahydro-3,7-dimethyl-8-[2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)ethyl]-1-naphthalenylester,[1S[1alpha(R*), 3alpha,7beta,8beta(2S*,4S*), 8abeta]]-, chemicalregistration no. 75330-75-5, marketed in several forms, e.g. Mevacor®from Merck & Co., Inc. Whitehouse, N.J., described in U.S. Pat. No.4,231,938 to Monaghan et al., November 1980, and G. S. Brenner et al.,in Analytical Profiles of Drug Stubstances and Excipients, vol 21, H.g.Brittain, Ed. (Academic Press, San Diego, 1992) pp 277-305.

[0081] 9) Mevastatin, Butanoic acid,2-methyl-,1,2,3,7,8,8a-hexahydro-7-methyl-8-[2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)ethyl]-1-naphthalenylester,[lS-[1-alpha(R*), 7-beta,8-beta(2S*,4S*), 8a-beta]]-, chemicalregistration no. 73573-88-3, marketed is several forms, e.g. Compactin®from from Merck & Co., Inc. Whitehouse, N.J., and described in U.S. Pat.No. 3,983,140 to Endo et al., September 1976 and reviewed in Endo, 1985,J Med Chem 28, 401-405.

[0082] Nonlipid Related Effects of Certain HMGCoA Reductase Inhibitors

[0083] Fluvastatin, simvastatin, and lovastatin (but not pravastatin)locally inhibit isoprenoid biosynthesis resulting in the followingantiatherosclerotic effects on the arterial wall: a) inhibition ofsmooth muscle cell migration and proliferation (reversed by mevalonate);b) fluvastatin and simvastatin also inhibit cholesterol esterificationand deposition induced by acetylated LDL in cultured macrophages(Corsini, et al., 1996). Simvastatin and lovastatin also reduce the rateof DNA synthesis and proliferation of a wide variety of cell types invitro, by inducing a cell cycle arrest in G1 (Addeo, et al., 1996;Wilcken, et al., 1997). This effect of simvastatin and lovastatin on DNAsynthesis arrest is antagonized by estrogen (Addeo, et al., 1996).

[0084] Inhibitors of Cholesterol Bile Acids Recycling:7-alpha-hyroxylase and relationship to HMG-CoA reductase

[0085] Cholesterol 7-alpha-hyroxylase and HMG-CoA reductase are locatednear each other on the endoplasmic reticulum. Newly synthesizedcholesterol seems to be the preferred substrate for cholesterol7-alpha-hyroxylase and its diurnal rise correlates with rise in enzymesynthesis. This enzyme is intricately linked with sterol synthesis andit's regulation (Danielsson and Sjovall, 1985). Therefore, it seemedlogical to assume that certain inhibitors of cholesterol-bile acidrecycling from the intestine may have cholesterol lowering effects thatwould also act to lower host tissue cholesterol available to parasites.

[0086] However, although bile acid binding drugs have not proven, todate, to be active used alone against parasites, these compounds mayenhance HMG-COA reductase inhibitor activity as demonstrated byHoogerbrugge, et al., 1990; Kuroda, et al., 1992; McTavish and Sorkin,1991; and, Wiklund, et al., 1993. This combination of an HMG-CoAreductase inhibitor plus a bile acid binding drug is likely be morepotent for antiparasitic therapy than any single HMGCoA reductaseinhibitor alone because of known enhanced anticholesteremic propertiesof the two drug types when administered together over either drug typegiven alone.

[0087] Medical concern that hypocholesteremics based on HMG-CoAreductase inhibition may result in untoward effects on nontarget tissuesdue to longterm physiologic consequences of depletion ofmevalonate-derived isoprenoids led to examination of cholesterolinhibition further down the synthetic path, at squalene synthesis.

[0088] Cytochrome P450 Enzyme Inhibitors: 14Alpha-Demethylase Inhibitionand Delta 24(25) Sterol Methyltransferase Inhibitors

[0089] The cytochrome P450 enzymes are a family of iron-containinghemoproteins. The P450 enzymes are generally divided based on structureand function. Those involved in steroidogenesis, the CYP11, CYP17,CYP19, CYP21 and CYP27 subfamilies; and in the metabolism of cholesteroland bile acids, the CYP7 and CYP51 subfamilies exhibit a high degree ofregio- and stereospecificity (Coon, et al., 1992; Mason and Hutt, 1997;Nebert, et al., 1991). Coincidentally, in evolutionary terms, thosecytochrome P450 enzymes involved in steroidogenesis are also the oldestmammalian P450's. Therefore, shared P450 steroidal enzymes are the mostlikely to be common to both humans and more primitive fungal orprotozoan parasites infecting humans. Therefore, drugs known tospecifically inhibit these P450 steroidal enzymes may also inhibitsimilar P450 enzymes of older, more primitive organisms.

[0090] Imidazole drugs, using ketoconazole as an example, may then haveantifungal/antiparasitic action for two reasons: (1) Direct action onparasite P450 steroidogenic enzymes reduces parasite de novo sterolsynthesis, particularly fungal and protozoal-specific ergosterolsynthesis via 14alpha-demethylase inhibition of lanosterol conversion toergosterol. (2) Also, indirectly because the human host intracellular orblood environment where the parasites must obtain cholesterol by“salvage” is likewise depleted of this second sterol required forleishmanial and trypanosomal survival.

[0091] Imidazoles can inhibit transformation of lanosterol to eitherErgosterol or Cholesterol (14alpha-) Imidazoles are typically considered“antifungals” for use in treatment of both superficial and systemicfungal infections (Heel, et al., 1982, Drugs 23, 1-28). However, variousother physiologic-drug effects with rising doses have resulted in use ofthese compounds for nonfungal indications. Examples of imidazolesinclude: ketoconazole, clotrimazole, aminoglutethimide, and etomidate.Doses and pharmacokinetics for imidazoles have been reviewed by Heel etal., 1982, supra.

[0092] The antifungal compound, ketoconazole, is believed to inhibitcholesterol biosynthesis via inhibition of the microsomal P-450 enzyme14alpha-demethylase. Additional known drug activities affectingsteroidogenesis of imidazoles in general and ketoconazol in particularinclude: (1) at therapeutic doses (200-600 mg/day) ketoconazole blockstestosterone synthesis in men (Feldman, 1986; Pont, et al., 1982) and athigh dose regimens caused substantial inhibition of testicular andadrenal steroidogenesis (Feldman, 1986); (2) ketoconazole blocks11beta-hydrolase and cholesterol side-chain cleavage for the adrenalsteroidogenic pathway (Feldman, 1986); (3) ketoconazole inhibits renal25-hydroxyvitamin D-24-hydroxyase (Vitamin D, an intracellular Ca⁺⁺regulator) (Feldman, 1986).

[0093] The actions of ketoconazole (as a representative imidazole)decreased human patient plasma cholesterol between 27% (at 1200 mg/day)to 15% (at 200 mg/day) from pretreatment baseline (Feldman, 1986).Ketoconazole and two other related 24(25) sterol methyltransferaseinhibitors were shown by Urbina, et al. (1995) to elucidate that24-alkyl sterols are essential growth factors for Trypanosoma cruzi andthat the preferred substrate of the delta 24(25) sterolmethyltransferase in this organism is zymosterol.

[0094] Miscellaneous Hypocholesteremics

[0095] 1) BERBERINES: The exception to these lipid cogener naturalantiparasitics are several natural and synthetic berberine/berbineanalogs (U.S. Pat. No. 5,290,553, to Iwu, et al., 1994). Berberineextracted from Coptis chinensis, lowered serum cholesterol level of micefed a high cholesterol diet (Chen and Xie, 1986) and is a knownhypocholesteremic. These natural and synthetic berberine/berbine analogshave been found to have potent antimalarial, antitrypanosomal, andantileishmanial properties (U.S. Pat. No. 5,290,553, to Iwu, et al.,1994)

[0096] 2) BETA-CAROTENE AND LYCOPENE are moderate hypocholesteremics.Fuhrman, et al. (1997, Biochem Biophys Res Commun 233, 658-662) reporteda 14% decrease in plasma LDL cholesterol, in humans given a dose of 60mg/day tomato lycopene for 3 months. In vitro, J-774 A.1 macrophages'cholesterol synthesis was inhibited 63% or 73% from acetate, but notfrom mevalonate, following treatement with 10 uM beta-carotene orlycopene, respectively (Fuhrman, et al., 1997, supra).

[0097] 3) ANTICANCER COMPOUNDS: In some cases, anticancer agents actbecause sterol synthesis in proliferating cells is ususally controlledby sterols that are produced intracellularly and is, independent ofextracellular cholesterol (Danielsson and Sjovall, 1985, Sterols andBile Acids. Elsevier, N.Y.). A linkage has been shown between de novocholesterol synthesis and is required for completion of the cell cycle(Bottomley, et al., 1980, FEBS Lett 119, 261-264). It would be expectedthat such anticancer agents (e.g. estrogen/testosteroneagonists/antagonists) would have some antiparasitic properties either byvirtue of lowering the cholesterol of the parasites' enviroment withinthe mammalian host (including man) or by direct inhibitory action on thesterol/cholesterol synthetic pathway of the parasites. It is well known(see above discussion) that while parasite and mammalian sterolmetabolic pathways differ in some basic fundamental steps, thesepathways for sterol production and incorporation share many commonsubstrates, enzyme cofactors, and result in the same products. Thus, itis not unreasonable to assume that an anticancer compound having a knownmode-of-action targeting a pathway common to both parasites and mammals(including man) would have fundamental and significant antiparasiteproperties.

[0098] One example is ketoconazole, which at moderate (200-600 mg/day)or high dose regimens inhibits both testicular and adrenalsteroidogenesis (Feldman, 1986, Endocrine Rev 7, 409-420). Examplesinclude: ketoconazole, clotrimazole, aminoglutethimide, and etomidate.At 400 mg/3×/day ketoconazole, prostate cancer subjects showed clinicalimprovement with few and minor side effects (Feldman, 1986, supra; Singhet al., 1995, J Assoc Physicians India 43, 319-320; Larbi et al 1995, AmJ Trop Med Hyg 52, 166-168; Trachtenberg, 1984, J Urol 132, 61;Trachtenberg, and Pont, 1984, Lancet 2, 433).

[0099] An second suitable example is tamoxifen,(Z)-2-[4-(1,2-Diphenyl-1-butenyl)phenoxy]-N,N-dimethylethanamine,(chemical registration no. 54965-24-1 and 10540-29-1, preparationdescribed in U.S. Pat. No. 4,536,516 to Harper et al., August 1985),best known as a nonsteroidal estrogen agonist for breast cancer adjuvanttherapy (Bryant and Dere, 1998, Proc Soc Exp Biol Med 217, 45-52; Majoret al., 1998, Orv Hetil 139, 121-124; Muller et al., 1998, Cancer Res58, 263-267). Among tamoxifen's known consequences is that it results inlowering of sterol synthesis and cholesterol levels in many bodytissues, including significant decreases in total serum and low densitylipoprotein (LDL) cholesterol levels, increase in high densitylipoprotein subclass 2 cholesterol, and increase in apolipoprotein A-I,a decrease in apolipoprotein B, and a reduction in serum concentrationof lipoprotein (a) in humans (Chang et al., 1996, Ann Oncol 7, 671-675;Elisaf et al., 1996, Anticancer Res 16, 2725-2728; Morales et al., 1996,Breast Cancer Res Treat 40, 265-270; Wasan et al., 1997, J Pharm Sci 86,876-879), and Wistar rats (Vinitha et al., 1997, Mol Cell Biochem 168,13-19). These effects on cholesterol may be due to a direct inhibitionof delta-8-isomerase (see Gylling et al., 1995, J Clin Oncol 13,2900-2905). A known side-effect during high-dose therapy (similar tocentral nervous system toxicty of antiestrogens of the clomiphene type)is cognitive impairment in 32% of patients, and 17% of standard-dosepatients, compared to 9% of control patients (van Dam et al., 1998, JNatl Cancer Inst 90, 210-218).

[0100] A third example is the estrogen antagonist, Raloxifene,Methanone,[6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thien-3-yl][4-[2-(1-piperidinyl)ethoxy]phenyl]-,(chemical registration no. 84449-90-1) which inhibits both coppermediated LDL oxidation as well as the cellular modification of LDL bymurine peritoneal macrophages. Raloxifene is a more potent inhibitor ofLDL oxidation than 17-beta-estradiol and (in rats) lowered cholesterollevels below control values within 4 days after initiation of treatment(Bryant and Dere, 1998, supra; Frolik et al., 1996, Bone 18, 621-627;Zuckerman and Bryan, 1996, Atherosclerosis 126, 65-75). Another estrogenantagonist, exemestane, 6-methyleneandrosta-1,4-diene-3,17-dione,chemical registration no. 107868-30-4, an irreversible inhibitor ofsteroidal aromatase, reduces total and HDL cholesterol and totaltriglyceride.

[0101] A fourth example is the antiestrogen, clomiphene citrate(Clomid®, Prepn: Allen et al., U.S. Pat. No. 2,914,563 in 1959 toMerrell); droloxifene/droloxifene citrate (Klinge Pharma, Germany) andZuclomiphene (=Transclomiphene, Marion Merrell Dow) which are also knowto have hypocholesteremic properties via inhibition of cholesterolbiosynthesis (Ke et al., 1997, Bone 20, 31-39; Ramsey and Fredericks,1977, Biochem Pharmacol 26, 1161-1167). Droloxifene was reported toreduce total serum cholesterol 65-70% compared to controls in rats (Keet al., 1995, Bone 17, 491-496). Similarly, toremifene (and tamoxifen)are reported to inhibit the conversion of delta-8-cholesterol tolathosterol so that total and LDL cholesterol levels are lowered bydownregulation of cholesterol synthesis. Thus, inhibition of thedelta-8-isomerase may be the major hypolipidemic effect of these agents(Gylling, et al., 1995, supra).

[0102] Many antiestrogens seem to work because estrogen is known toelevate plasma cholesterol concentration (Klimis-Tavantzis et al., 1983,J Nutr 113, 320-327); However, the disadvantage is that these also seemto lower cholesterol biosynthesis in the central nervous system andneurotoxic effects are known for many antiestrogens including theclomiphe

[0103] ne and derivatives (Ramsey 1978, Biochem Pharmacol 27,1637-1640).

[0104] Other possible antiparasitic/antifungal compounds include:

[0105] Thyroid hormone antagonists, suitable examples includedextrothyroxine,D-4-(4-Hydroxy-3,5-diiodophenoxy)-3,5-diiodobenzylalanine;0-(4-hydroxy-3,5-diiodophenyl)-3,5-diiodo-, or D-thyroxine, chemicalregistration no. 51-49-0, and dextrothyroxine sodium, chemicalregistration no. 7054-08-2 and 137-53-1, marketed as Choloxin® by KnollPharmaceutical Co. Mount Olive, N.J., U.S. Pat. No. 2,889,363 to Gingeron June 1959.

[0106] Antihyperlipoproteinemic agents which inhibit cholesterolreabsorption as bile acids. Suitable examples include cholestyramineresin, chemical registration no. 58391-37-0 or 11041-12-6 (Ast andFrishman, 1990, J Clin Pharmacol 30, 99-106), marketed in several forms,e.g. Questran® from Bristol-Myers Squib, Wallingford, Conn. In the samecategory is colestipol, chemical registration no. 50925-79-6 orcolestipol hydrochloride,1,2-Ethanediamine,N-(2-aminoethyl)-N′-[2-[(2-aminoethyl)amino]ethyl]-,polymer with (chloromethyl)oxirane, chemical registration no.37296-80-3, preparation described in U.S. Pat. No. 3,803,237 to Ledniceret al., April 1974, reviewed in Heel et al., 1980, Drugs 19, 161-180,and marketed as Cholestid® from Pharmacia and Upjohn, Inc. Kalamazoo,Mich.

[0107] Antihyperlipoproteinemics, suitable examples include:

[0108] clofibrate, Propanoic acid, 2-(4-chlorophenoxy)-2-methyl-, ethylester, chemical registration no. 637-07-0, described in Hassan andElazzouny, 1982, Anal Profiles Drug Subs 11, 197-224, marketed inseveral forms, e.g. Atromid-S® from Wyeth-Ayerst, Philadelphia, Pa.

[0109] Antihyperlipoproteinemics which inhibit synthesis of VLDL,possibly by inhibiting synthesis of ApoB-100), for example, Gemfibrozil,Pentanoic acid, 5-(2,5-dimethylphenoxy)-2,2-dimethyl-; Valeric acid,2,2-dimethyl-5-(2,5-xylyloxy)-, chemical registration no. 25812-30-0,preparation described in U.S. Pat. No. 3,674,836 to Creger on July,1972, marketed in several forms, e.g. Lopid® from Parke-Davis, MorrisPlains, N.J.

[0110] Antihyperlipoproteinemics which inhibit synthesis of cholesteroland increase fecal excretion of bile acids, and may decrease plasma HDLlevels, e.g. Probucol, Acetone, bis(3,5-di-tert-butyl-4-hydroxyphenyl)mercaptole;4,4′-[(1-methylethylidene)bis(thio)]bis[2,6-bis(1,1-dimethylethyl)phenol],chemical registration no. 23288-49-5, preparation described in U.S. Pat.No. 3,576,883 to Neuworth, M. B. on April, 1971, and its use as acholesterol-lowering agent in U.S. Pat. No. 3,862,332 to Barnhart etal., on January 1975, marketed in several forms, e.g. Lorelco®, byHoechst Marion Roussel, Inc. Kansas City, Mo.

[0111] Antihyperlipoproteinemics which inhibit cholesterol lumenalabsorption resulting in reduced serum LDL and serum cholesterol(Morehouse et al., 1999, J Lipid Res 40, 464-474. For example,Tiqueside, beta-D-Glucopyranoside, (3beta,5alpha,25R)-spirostan-3-yl4-O-beta-D-glucopyranosyl- chemical registration no. 99759-19-0, andPamaquesideSpirostan-1]-one,3-[(4-O-beta-D-glucopyranosyl-beta-D-glucopyranosyl)oxy]-,(3beta,5alpha,25R)—; (2) 11-Oxo-(25R)-5alpha-spirostan-3beta-yl4-O-beta-D-glucopyranosyl-beta-D-glucopyranoside, chemical registrationno. 150332-35-7, both available from Pfizer Laboratories, New York, N.Y.

[0112] Inhibitors of type II fatty acid synthesis such as cerulenin,2,3-Epoxy-4-oxo-7,10-dodecadionamide. Antifungal antibiotic isolatedfrom several species, including Acremonium (Cephalosporium),Acrocylindrum, and Helicoceras. It inhibits the biosynthesis of severallipids by interfering with enzyme function, chemical registration no.17397-89-6, preparation described in Boeckman and Thomas, 1977, (J AmChem Soc 99, 2805).

[0113] Antineoplastic agents, suitable examples including Ifosfamide,N,3-bis(2-chloroethyl)tetrahydro-2H-1,3,2-Oxazaphosphorin-2-amine,2-oxide;3-(2-chloroethyl)-2-[(2-chloroethyl)amino]tetrahydro-1,3,2-Oxazaphosphorine,2-oxide, chemical registration no. 3778-73-2, preparation described inU.S. Pat. No. 3732,340 to Arnold et al. in May, 1973, reviewed inSchoenike and Dana, 1990, Clin Pharm 179-191, marketed in several forms,e.g. Ifex® from Bristol-Meyers Oncology Division, Princeton, N.J.

[0114] Anticholelitholytic agents such as monoctanoin, Octanoic acid,monoester with glycerol, Octanoic acid, monoester with1,2,3-propanetriol, chemical registration no. 26402-26-6, preparationand use described in U.S. Pat. No. 4,205,086 to Babayan, May, 1980,marketed as Monoctanin® from Ethitek Pharmaceuticals Co., Skokie, Ill.

[0115] The compounds of the invention can be assayed by any techiquesknown in the art in order to demonstrate their antiparasitic/antifungalactivity. Such assays include those described below in the Materials andMethods. Those compounds which are demonstrated to have significantantiparasitic/antifungal activity can be therapeutically valuable forthe treatment or prevention of leishmania, trichomoniasis, andtrypanosomiasis.

[0116] Pharmaceutical compositions comprising the inhibitive compoundsor the salts thereof are provided by the present invention.Administration of these compositions include, but are not limited to,oral, intradermal, transdermal, topical, mucosal, intravenous,subcutaneous, intramuscular, intraperitoneal, and intranasal routes.More than one administration to the patient may be necessary. Theoptimum amount of the antiparasitic/antifungal agent varies with theweight of the patient being treated, with some amount ranges presentedin the patents describing these agents. A range includes dosages of 0.1mg/Kg/day to 50 mg/Kg/day.

[0117] A further embodiment of this invention includes the combinedtherapy that can be obtained by treating patients with parasitic orfungal diseases with a combination of the compounds of this invention.The combination is preferably chosen such that the inhibitory activityof the combined compositions is different, i.e. the pathway is blockedat different points. The efficacy of combined treatment could besubstantially better than one composition alone due to the ability tomodulate different effects of the compounds and possibly reducingside-effects or toxicity. The administration of the compounds in thecombination could be simultaneous or sequential or in different doseforms including combinations of oral dose forms with injectables to namejust a few.

[0118] The invention can be better understood by referring to thefollowing examples which are given for illustrative purposes only andare not meant to limit the invention.

[0119] The following MATERIALS AND METHODS were used in the examplesthat follow.

[0120] Trypanosome (a) IN VITRO drug screening method: 14 clinicalisolates of Trypanosoma brucei rhodesiense (agent of East AfricanSleeping Sickness) many of which are refractory to standard trypanocidessuch as diamidines and melarsoprol (Bacchi et al., 1990, AntimicrobAgents Chemother 34, 1183-1188) are maintained as laboratory infectionsin rats and mice and as frozen stabilates of blood forms. Six of theseisolates have been cultivated as blood forms in modified Iscove's medium(Hirumi & Hirumi, 1989, J Parasitol 75, 985-989). These cultivated bloodforms are fully infective and serve as the initial screen fortrypanocides based on a 24 well culture plate system and readings doneon duplicate wells using a Coulter Counter (Bacchi and Yartlett, 1993,Acta Tropica 54, 225-236; Bacchi et al., 1996, Antimicrob AgentsChemother 4, 1448-1453). This screen has proven highly reproducible,needs minimal drug since the volume is limited (1 ml medium/well) and iscomplete after 48 h. It has proven to be highly predictive of in vivoactivity (e.g., Brun et al., 1996, Antimicrob Agents Chemother 40,1442-1447; Bacchi et al., 1996, supra).

[0121] Usually plant extracts are screened vs. four isolates, T. b.brucei Lab 110 EATRO (veterinary parasite, and 3 strains of T. b.rhodesiense (KETRI 243, KETRI 243 As10⁻³ and KETRI 269). The latter arehuman clinical isolates refractory to arsenicals and diamidines (KETRI243 and 243 As10-3) or DFMO KETRI 269 (Bacchi et al., 1990, supra).Extracts giving IC50 values at or below 100 ug/ml will be considered forfurther testing, using a more purified extract.

[0122] Trypanosome IN VIVO drug screening method: Extracts havingsignificant activity in vitro (IC50 <10 ug/ml) along with reasonableevidence of selectivity in mammalian toxicity tests will be studied inin vivo screens. Initially the standard T. b. brucei Lab 110 EATRO mousemodel will be used. Agents proving active in this model will be testedin the T. b. rhodesiense KETRI 243 and 269 model infections. Thesescreens have proven effective in identifying in vivo trypanocidalactivities of DFMO and other polyamine analogs, nuceloside analogs,arylguanylhydrzones and other agents (e.g., Bacchi et al., 1980, Science210, 332-334; 1987, Antimicrob Agents Chemother 31, 1406-1413; 1990,supra; 1992, Antimicrob Agents Chemother 35, 2736-2740; 1997, AntimicrobAgents Chemother 41, 2108-2112). Usually compounds are given by i.p.injection once daily for 3 days. Animals surviving >30 days beyond thedeaths of untreated, infected controls with no evidence of bloodparasites are considered cured. Other routes used include i.v., per osand Alza mini osmotic pumps which are implanted under the skin andrelease 1 ?l of drug solution per h for 3 or 7 days. Agents or extractsproving active in the above screens will be studied in a standard CNSmodel infection (Jennings et al., 1983, Contrib Microbiol Immunol 7,147-154) which we have used to demonstrate efficacy of single agents ordrug combinations (e.g., Clarkson et al., 1983, Proc Natl Acad Sci 80,5729-5733; Bacchi et al., 1987, supra; 1996, supra), This model takes10-12 months to resolve and will only be used for highly active extractsand purified substances.

[0123] TRICHOMONAD IN VITRO AND IN VIVO DRUG SCREENING METHODS.(a) INVITRO: Several strains of T. vaginalis are maintained, covering thespectrum of metronidazole resistance: CDC85 (highly resistant); RU384,RU383, IR78 (moderately resistant); NYH286, RU284, RU393, C1-NIH(sensitive). These strains are routinely cultivated in a non-definedmedium incorporating tryptose, yeast extract, maltose and supplementedwith 10% horse serum.

[0124] In in vitro drug studies, plant extracts are tested using a96-well plate assay as described by Meingassner et al., (1978,Antimicrob Agents Chemother 13, 1-3). This method uses only 200 ul ofmedium/well and thus uses very little plant extract. Results (intriplicate) are presented as “Minimal Inhibitory Concentration” (MIC)the lowest concentration completely blocking growth (Meingassner et al.,1978, supra). This method is also useful in comparing susceptibility ofvarious strains (Meingassner et al., 1978, supra; Yarlett et al., 1987,Mol Biochem Parasitol 24, 255-261). Assays will initially be done buthighly active compounds will also be tested anaerobically, sincemetronidazole resistance is only detectable under aerobic assayconditions (Meingassner et al., 1978, supra). Resistance tometronidazole is proposed to be due to the presence of defective oxygenscavenging mechanisms and resulting redox cycling of the partly reduceddrug (Yarlett et al., 1986, Mol Biochem Parasitol 19, 111-116). Sincevaginal O₂ tensions are {fraction (1/20)}th to ¼th of atmospheric(Wagner et al., 1978, Fertil Steril 30, 50-53), it is more physiologicalto do drug sensitivity testing under conditions approaching this.

[0125] (b) IN VIVO: Extracts or highly purified material proving activein vitro (MIC 0.5 mg/ml) with favorable selectivity will also be testedin vivo in a mouse subcutaneous infection model which has been used tocorrelate virulence of T. vaginalis isolates with severity ofpathogenicity in the human host (Honigberg et al., 1966, Acta Cytol 10,353-361; Kulda et al., 1970, Am J Obstet Gynecol 108, 908-918). Thismodel has been used successfully to test various agents fortrichomonacidal activity and is considered superior to other in vivotests (Brenner et al., 1987; Kulda, 1989, Trichomonads Parasitic inHumans. Springer-Verlag, New York, pp 112-154).

EXAMPLE 1

[0126] Nineteen plant extracts were examined for activity in vitroagainst four strains of animal or human-pathogenic African trypanosomes,and three strains of mammalian-pathogenic Trichomonas spp.

[0127] The trypanosomes studied were Trypanosoma brucei brucei Lab 110EATRO, which is pathogenic to cattle and other livestock, and severalstrains of Trypanosoma brucei rhodesiense, a parasite of humans,domestic and wild animals. Strains of T. b. rhodesiense included drugresistant clinical isolates KETRI 243 and 269 and KETRI 243 As-10-3, ahighly melarsen- and diamidine-resistant clone of KETRI 243. The 19extracts were tested in an in vitro screen using a semi-defined mediumfor growth of bloodstream trypomastigotes at 37° C. (Hirumi & Hirumi,1989, supra) to determine IC₅₀ values (Bacchi et al., 1996, supra).Using a cutoff of 100 ug/ml, 12 of the 19 extracts consistently gaveIC₅₀ values in the active range (Table 1). Of these, 10 had IC₅₀ valuesat or below 10 ug/ml and were considered sufficiently active to warranttesting of more purified extracts. TABLE 1 Activity of plant extracts vsgrowth of African trypanosomes in vitro. Bloodform trypanosomes weregrown in 24 well culture dishes (1 ml/well) in HMI-18 medium (Hirumi &Hirumi, 1989, supra). One half of the culture volume was replaced dailywith fresh medium plus drug. Each extract was dissolved in 100% DMSO anddiluted with medium. Cells were counted daily with a coulter counter.Data are as IC₅₀ values in ug extract/ml culture. Four strains wereused: T.b. brucei Lab 110 EATRO, and three T.b. rhodesiense clinicalisolates from the Kenya Trypanosomiasis Research Institute (KETRI). Alldata from 48 hr cultures. Control cell counts averaged 5 × 10⁶ cell/mlat 48 h. IC₅₀ (ug/ml) KETRI 243 EATRO 110 KETRI 243 KETRI 269 As-103SU-367 9.2 15.1 8.4 8.5 SU-369 11 5.1 8.2 11 SU-370 64 5 500 ug/ 500 ug/ml-22% ml-22% SU-766 102 21.5 500 ug/ 47 ml-22% SU-787 9.0 8.5 12.5 14.9SU-813 500 ug/ 500 ug/ 500 ug/ 500 ug/ ml-38% ml-14% ml-44% ml-22%SU-614 134 74 79 51 SU-105 500 ug/ 500 ug/ 500 ug/ 500 ug/ ml-16% ml-8%ml-7% ml-8% SU-719 1.9 2.0 1.6 3.4 SU-679 18.0 19.5 28.9 40.5 SU-799 115229 114 117 SU-740 33 32.5 30.0 39.0 SU-175 6.5 5.4 6.8 6.2 SU-847 13.58.3 12.5 12.6 SU-848 14.1 16.0 18.0 15.1 SU-769 119 73.0 74 78 SU-7246.4 64.0 59 105 Pentamidine 0.00048 0.00186 0.00192 0.003 Melarsen0.00077 0.0025 0.0066 0.0072 Oxide

[0128] Several secondary extracts were recently shipped and tested(Table 2), while others are being prepared. Of the four secondaryextracts supplied, one, SU1460, derived from the primary extract SU787of Aframomum aulocacarpus, featured a 10-15 fold increase in activity.SU787 had 1C50 values of 8.5-14.9 ug/ml (Table 1), while the value forSU1460 was 0.86 ug/ml. TABLE 2 Activity of Secondary Plant Extracts onAfrican Trypanosomes. Assay method as in Table 1. Results as IC₅₀ inug/ml. Primary Secondary IC₅₀ Extract Origin Extract T.b.brucei 110SU-724 Araliopsis tabouensis AT6 SU-1459 500*    SU-724 Araliopsistabouensis AT7 SU-1458 100*    SU-787 Aframomum aulocacarpus AZ₂ SU-14600.86 SU-175 Dracaena mannii SU-1461 6.4 Mannispirostan A

[0129] Eight additional primary extracts were also tested in thetrypanosome screen (Table 3). Of these, SU1462 from Napoleonaeaimperialis and SU1464 from Glossocalyx brevipes were highly active(IC₅₀˜1 ug/ml) and warrant further study. TABLE 3 Growth inhibitoryactivity of new primary plant extracts against african Trypanosomes.Assay method as in Table 1. Results as IC₅₀ in ug/ml. IC₅₀ ExtractOrigin T.b. brucei 110 SU 1462 Napoleonaea imperialis MeOH 1.75 SU 1463Pachypodanthium staudtii CH₂Cl₂ 88 SU 1464 Glossocalyx brevipes CH₂Cl₂0.77 SU 1465 Enantia chlorantha MeOH 10.5 SU 1465 Eupatorium odoratumMeOH EOO 30% @ 50 ug/ml* SU 1467 Cleistopholis patens EtOH 62 SU 1468Leidobotrys staudii CH₂Cl₂ SU 1469 Ancistrocladus bateri ABSBM 28

[0130] The trichomonad screen consists of two human pathogenicTrichomonas vaginalis strains and a livestock parasite Tritrichomonasfoetus. The T. vaginalis isolates include a metronidazole sensitiveisolate (C1-NIH: ATCC 30001) and a strain highly resistant tometronidazole (CDC-085: ATCC 50143). The sceening procedure used is thatof Meingassner et al. (1978, supra) and determines the minimalinhibitory concentration (MIC) in mg/ml needed to completely inhibitgrowth. Table 4 detail data from the initial group of 19 primaryextracts. Of these, seven had MIC values of 1 mg/ml for all threeisolates and were considered of interest for further study. The resultsto Dec. 31, 1997 appear in Table 5. The most active extract in thisgroup was SU1464 from Glossocalyx brevipes which had an MIC value of0.0125 mg/ml for each isolate and was the most potent of the primaryextracts tested thus far. TABLE 4 Minimum inhibitory concentration(mg/ml) of plant extracts against Trichomonas vaginalis strain C1-NIH(ATCC#30001) susceptible to current drug therapy (metronidazole andCDC-085 (ATCC#50143) resistant to metronidazole therapy; and the cattleparasite Tritrichomonas foetus KV-1. Assays were performed in 200 ulmultiwell plates (96 well) by serial dilution of each compound (2.5 to0.0012 mg/ml final concentration) and inoculated with 6.6 × 104 cells.Plates were scored after 48 h according to motility (4 = 100%; 0 = nomotility) compared to control wells lacking the test compound(Meingassner et al., .1978, supra). MIC (mg/ml) C1-HIH CDC-085 KV-1ICBG# 48 hrs 48 hrs 48 hrs SU-105 >2.50 2.50 >2.50 SU-175 2.50 2.50 2.50SU-367 12.50 12.50 0.78 SU-369 0.62 1.25 1.25 SU-370 2.50 2.50 2.50SU-614 1.25 0.62 1.25 SU-679 0.62 0.62 0.62 SU-719 0.31 0.01 0.15 SU-7240.62 0.62 2.50 SU-740 1.25 1.25 1.25 SU-766 1.25 1.25 2.50 SU-769 0.310.62 0.62 SU-787 0.62 1.25 2.50 SU-798 1.25 0.62 1.25 SU-799 0.15 0.310.62 SU-813 >2.50 >2.50 0.15 SU-846 2.50 1.25 2.50SU-847 >2.50 >2.50 >2.50 SU-848 2.50 2.50 2.50 Metronidazole 0.003 0.400.004

[0131] TABLE 5 Inhibition of Trichomonas growth by new plant extracts.Assay method as in Table 4. Data expressed as MIC in mg/ml. ND, notdetermined. MIC Extract Origin C1-NIH CDC-085 KV1 SU 1463Pachypodanthium 0.80 ND >0.80 staudtii CH₂Cl₂ SU 1464 Glossocalyx 0.01250.0125 0.0125 brevipes CH₂Cl₂ SU 1465 Enantia 0.80 ND 0.40 chloranthaMeOH SU 1467 Cleistopholis >0.80 0.10  >0.80 patens EtOH SU 1468Leidobotrys 0.40 ND >0.80 staudii CH₂Cl₂ SU 1469 Ancistrocladus 0.40 ND0.40 bateri ABSBM Metronidaxole 0.003 0.40  0.003

[0132] The most active primary plant extracts in each screen are listedin Table 6. These were chosen on the basis of MIC levels (<1 mg/ml) fortrichomonad screens and IC₅₀ values (</=10 ug/ml) for trypanosomalscreens. Although many of the extracts were most active only against onegroup of organisms, six primary extracts had significant activityagainst both groups. These were SU369, 719, 724, 787, 1464 and 1465. Ofthese, SU719 and 1464- appeared to be most potent in both screens. TABLE6 Most active: ICBG primary plant extracts. Trichomonas Trypanosomes(MIC < 1 mg/ml) (IC₅₀ ≦ 10 ug/ml) SU-369+ SU-175** SU-679* SU-367SU-719*+ SU-369+ SU-724+ SU-719**+ SU-769* SU-724+ SU-787+ SU-787**+SU-799* SU-798** SU-1464*+ SU-846  SU-1462 SU-1465+ SU-847  SU-1464**+SU-1469 SU-848  SU-1465+

[0133] Although large-scale testing of plant extracts for activityagainst protozoan parasites is largely lacking (Wright & Phillipson,1990, Phytotherapy Res 4, 127-139) recent evaluation of Africanmedicinal plants vs. T. b. rhodesiense has given some encouragingresults (Freiburghaus et al. 1996a, J Ethnopharmacol 55, 1-11; 1996b,Trop Med Int Health 1, 765-771; 1997, Acta Tropica 66, 79-83). In thesestudies crude extracts were considered to have promising activity in anin vitro screen against blood forms if IC₅₀ values were at or below 10ug/ml. In the above trypanosome screen 13 of 27 primary medicinal plantextracts had such activity while two (SU719 and 1464) had IC₅₀ values ator below 1 ug/ml. Further studies will need to examine the selectivityof active extracts, i.e. the maximum tolerated concentrations bymammalian cell lines vs. the IC₅₀ or MIC values. If the selectivity datais favorable, further purification of the active principles and animaltesting would be the logical next steps in the exploration of theseextracts.

EXAMPLE 2

[0134] Using the leishmanial in vitro radiorespirometric bioassay theactive compound was purified and its structure determined. A relatedspecies, Aframomum meleguata, showed moderate activity againstTrypanosoma brucei in vitro IC₅₀ 9.0 ug/ml. However, a third plantspecies, Aframomum aulocacarpus, showed activity within the highlyactive drug range, IC₅₀ 0.86 ug/ml, a 10-11-fold increase in activity.The structural modifications in active antiparasitic with thesebotanical species changes are in progress.

EXAMPLE 3

[0135] Numerous similarities in leishmanial and trypanosomal lipiduptake and metabolism may explain common natural product drugsusceptibility. Inhibitor, of cholesterol synthesis, metabolism, and/orexcretion described above in the detailed description were tested versustrypanosome isolates grown as bloodforms in HMI-18 medium containing 10%fetal bovine serum. Coulter counds were made daily and IC50 valuesdetermined after 48 h. Results are shown in Table 7. TABLE 7 Drugcompounds tested vs trypanosome isolates. Lab IC₅₀ (ug/ml) 110 EATRO 243269 243 As 10-3 General inhibitors Atromid-S >100 >100 — —Lopoid >100 >100 — — Bile Acid resorption inhibitorsCholestipol >100 >100 — — Questran >100 >100 — — HMG-CoA reductaseinhibitors Baycol 13 7.7 — 52 Nevacor 3.3 4.4 6.9 — Pravachol >100 >100— — Zocor 1.33 12.9 7.0 — Lescol >100 >100 — — Hormoneagorlists/antagonists Tamoxifen 30 Citrate* Tamoxifen* 27 Squaleneoxidase inhibitors Lamisil 1.3 86 77 >100 Thyroid hormone antagonistsCholoxin >100 >100 — —

EXAMPLE 4

[0136] Targeted Anti-lipid Antileishmanials for Specialized Testing inPrimates.

[0137] Two compounds selected as inhibitors of cholesterol synthesisand/or metabolism, and/or excretion will be tested at 3 dose levels inmonkeys for evaluation against both monkey cutaneous and monkey visceralleishmaniasis. In 4 experiments we want to test 2-drug-combinations (4combinations) as antileishmanials. The combinations we propose arealready given in combination (for nonleishmanial indications) to humans.These drug combinations studies are in progress:

[0138] Positive Control Drug (Glucantime-Treated) Animals: (IPAdministration)

[0139] dose 1-13 mg/kg/day (MKD)

[0140] dose 2-52 mg/kg/day

[0141] dose 3-104 mg/kg/day

[0142] Negative Control (Suspending Drug Vehicle-HEC Tween Minus Drug):(IP Administration)

[0143] PO Administration:

[0144] Drug 1 dose 1 (*human dose MKD level)

[0145] Drug 1 dose 2 (10× human dose MKD level)

[0146] Drug 1 dose 3: (10OX human dose MKD level)

[0147] Drug 2 dose 1 (*human dose MKD level)

[0148] Drug 2 dose 2 (10× humand dose MKD level)

[0149] Drug 2 dose 3: (10OX human dose MKD level)

[0150] Drug 1 dose 1+drug 2 dose 1

[0151] Drug 1 dose 2+drug 2 dose 1

[0152] Drug 1 dose 3+drug 2 dose 1

[0153] Drug 1 dose 1+drug 2 dose 2

[0154] Drug 1 dose 2+drug 2 dose 2

[0155] Drug 1 dose 3+drug 2 dose 2

[0156] Drug 1 dose 1+drug 2 dose 3

[0157] Drug 1 dose 2+drug 2 dose 3

[0158] Drug 1 dose 3+drug 2 dose 3

[0159] Candidate drugs 1 & 2 Vehicle Control (corn oil)

[0160] *Dose will vary depending on the drug being tested.

[0161] Discussion

[0162] Cholesterol is a sterol regulating the membrane fluidity ofeukaryotic membranes (Stryer, 1988, Biochemistry. WH Freeman andCompany, New York). Cholesterol contains a bulky steroidal nucleus witha hydroxyl group at one end and a flexible hydrocarbon tail at the otherend (FIGS. 12-29, Stryer, 1988, supra). Cholesterol inserts intomembrane lipid bilayers so that the hydrocarbon tail is located in thenonpolar core with the hydroxyl group bound to a carbonyl oxygen atom ofa phospholipid polar head group oriented toward the aqueous exterior orinterior of the cell (model previous page). The interaction forcesbetween sterol molecules seem to be little affected by the double bondin the ring system or modifications in the side chain. Also, the changein orientation of the hydroxyl group from 3-beta to 3-alpha does notsignificantly alter the cross-sectional area of the sterol at thesurface. However, replacement of the hydroxyl group by an oxogroup, orchanges in the planar structure of the sterol nucleus, increase themolecular area, and may lead to some degree of membrane destabilization.This is why certain dimerized natural product plant components haveantiparasite properties. Cholesterol prevents the crystallization offatty acid chains by fitting between them. Thus, high concentrations ofcholesterol tend to abolish phase transitions of lipid bilayers (Bloch,1983, CRC Critical Reviews in Biochemistry 14, 47-92). Cholesterol (andsterol)-mediated stabilization from phase transitions of lipid bilayersis undoubtedly critical to the survival of Kintoplastida parasites whichmust undergo marked temperature transition from ambient (within theinsect vector) to mammalian body temperature (37° C. or greater,dependent on reservoir or human mammalian host) during their life cycle.Dependent on Tm, melting temperature, fatty acid acyl chains in bilayerscan exist either a more rigid or ordered state favoring trans C—C bonds;or, at rising temperature, a more disordered or gauche C—C bondconformation (a 120-degree rotation, clockwise, g+, or counterclockwise,g−) increases. The transition temperature, Tm, depends upon the lengthof the fatty acyl chains and amount of unsaturation. Saturated fattyacids result in an elevated Tm (e.g. Crisco shortening, a solid at roomtemperature) whereas, greater unsaturation increases fluidity (e.g.vegetable oils, liquid at room temperature) lowering Tm. Likewise,cholesterol prevents rigidity (crystalization) by fitting in betweenfatty acids increasing fluidity, so that at high membrane cholesterolconcentrations, phase transition of bilayers are largely abolished. Anopposite effect of cholesterol is to sterically block large motions offatty acyl chains, making membranes less fluid. Membrane fluidity, i.e.cholesterol content therefore, and indeed sterol content in general, hasstrigent biologic control for each cell type/function (Thompson, 1992,The Regulation of Membrane LiDid Metabolism, CRC Press, Ann Arbor).

[0163] Medicinal herbs are of considerable importance to the health ofindividuals and communities worldwide. Even in industrialized countries,an estimated 33% of the population use alternative treatments includingherbal remedies. Approximately 35,000 to 70,000 plant species have beenused for medical purposes (Zhang, 1996, World Health 49th year(2):4-5).Given the extraordinary ratio (approaching 50%) of “active to totalscreened” plants developed from our ICBG ethnomedical and ethnobotanical“leads” for antiparasitics, one must be impressed by the accuracy of thetraditional healers' information. The fact that in the United States,two thirds of the drugs currently available on the market are originallybased on medicinal plants then becomes somewhat less astounding(Micozzi, 1996, World Health 49th year (2):8-9). Most currentantimalarials and other trypanosomals have their chemical origins inherbal extracts, thus, scientific history would lead one to believe thatour ICBG approach is scientifically justified. The data presented inthis disclosure support the that conclusion that the herbal extractswhich, in fact chemically resemble various components of sterolbiosynthesis and metabolism, act by inhibition of this pathway. Themarked antiparasite efficacy of the known anticholesterol,antihyperlipidemics, cholesterol hormone antagonists, and anticancerdrugs affecting this pathway for 3 of the four human parasite genera wehave studied to date, not only provides immediate new chemotherapy forthese infections in man and animals, but supports the concept thatefficacious and nontoxic therapy for these diseases will be based oncompounds affecting this pathway.

What is claimed is:
 1. An antiparasitic composition comprising aninhibitor of cholesterol synthesis.
 2. An antiparasitic compositioncomprising an inhibitor of cholesterol metabolism.
 3. An antiparasiticcomposition comprising an inhibitor of cholesterol excretion.
 4. Anantiparasitic composition comprising at least one inhibitor selectedfrom the group consisting of inhibitor of cholesterol synthesis,inhibitor of cholesterol metabolism, and inhibitor of cholesterolexcretion.
 5. A method for treating an individual including a human witha parasitic infection comprising administering to said individual acholesterol synthesis inhibitor in a pharmaceutically effective amount,in a pharmaceutically effective excipient.
 6. A method for treating anindividual including a human with a parasitic infection comprisingadministering to said individual a cholesterol metabolism inhibitor in apharmaceutically effective amount, in a pharmaceutically effectiveexcipient.
 7. A method for treating an individual including a human witha parasitic infection comprising administering to said individual acholesterol excretion inhibitor in a pharmaceutically effective amount,in a pharmaceutically effective excipient.
 8. A method for preventingparasitic infection in an animal including a human comprisingadministering to said animal a cholesterol synthesis inhibitor in apharmaceutically effective amount, in a pharmaceutically effectiveexcipient.
 9. A method for preventing parasitic infection in an animalincluding a human comprising administering to said animal a cholesterolmetabolism inhibitor in a pharmaceutically effective amount, in apharmaceutically effective excipient.
 10. A method for preventingparasitic infection in an animal including a human comprisingadministering to said animal a cholesterol excretion inhibitor in apharmaceutically effective amount, in a pharmaceutically effectiveexcipient.
 11. A method according to claim 5 wherein said administrationis selected from the group consisting of oral, topical and parenteral.12. A method according to claim 6 wherein said administration isselected from the group consisting of oral, topical and parenteral. 13.A method according to claim 7 wherein said administration is selectedfrom the group consisting of oral, topical and parenteral.
 14. A methodaccording to claim 8 wherein said administration is selected from thegroup consisting of oral, topical and parenteral.
 15. A method accordingto claim 9 wherein said administration is selected from the groupconsisting of oral, topical and parenteral.
 16. A method according toclaim 10 wherein said administration is selected from the groupconsisting of oral, topical and parenteral.
 17. An antiparasiticcomposition comprising an agent which interferes with parasitecholesterol uptake.
 18. An antiprasitic composition comprising aninhibitory agent selected from the group consisting of inhibitors ofcholesterol production in host, inhibitors of cholesterol transport tomonocyte of host, inhibitors of cholesterol delivery to the parasite,inhibitors of choline production, inhibitors of HMG-COA reductase,inhibitors of squalene oxidase, inhibitors of squalene synthetase,inhibitors of 14alpha-demethylase, inhibitors of bile acids resorption,inhibitors of butyrate, anticance hormone agonists/antagonists,hypocholesteremics, or a combination thereof.
 19. An antiparasiticcomposition comprising one or more hypocholesteremic.
 20. Theantiparasitic composition of claim 19 wherein the hypocholesteremic isbeta-carotene or lycopene.
 21. The antiparasitic composition of claim 19wherein the hypocholesteremic is an estrogen agonist or antagonist. 22.A method for treating an individual with a parasitic infectioncomprising administering to said individual an antiparasitic compositionof claim 18 in a pharmaceutically effective amount, in apharmaceutically effective excipient.
 23. A method for preventingparasitic infection in an individual comprising administering to saidindividual an antiparasitic composition of claim 18 in apharmaceutically effective amount, in a pharmaceutically effectiveexcipient.
 24. A method according to claim 22 wherein saidadministration is selected from the group consisting of oral, topical,and parenteral.
 25. An antiparasitic composition comprising anantiparasitic effective amount of at least one compound which inhibits aparasite's cholesterol biosynthesis, metabolism, and/or excretion. 26.An antiparasitic composition comprising an antiparasitic effectiveamount of a combination of compounds selected from the group consistingof inhibitors of cholesterol production in host, inhibitors ofcholesterol transport to monocyte of host, inhibitors of cholesteroldelivery to the parasite, inhibitors of choline production, inhibitorsof HMG-CoA reductase, inhibitors of squalene oxidase, inhibitors ofsqualene synthetase, inhibitors of 14alpha-demethylase, inhibitors ofbile acids resorption, inhibitors of butyrate, anticance hormoneagonists/antagonists, hypocholesteremics,